Media combinations and methods for establishing a regeneration system in zantedeschia hybrids using tubers

Abstract

The present application relates to a kind of culture medium combination and its method for building color calla regeneration system using tuber, and the culture medium combination includes: callus induction medium, proliferation medium, differentiation medium, wherein, induction medium: with MS as base medium, also contain: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L;Or, also contain the following concentration of substance: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L.The present application uses the aseptic budless tuber of color calla grown to specific period as explant, is combined with specific induction medium, proliferation medium and differentiation medium to obtain multiple shoots, and the method is high in callus induction rate, and it provides technical support for color calla tissue culture rapid propagation and the establishment of genetic transformation system.

Description

Technical Field

[0001] This invention belongs to the field of constructing a regeneration system for colored calla lilies, and specifically relates to a culture medium combination and method for constructing a regeneration system for colored calla lilies using tubers. Background Technology

[0002] Currently, the industrialized production of colored calla lily bulbs relies on rapid propagation through tissue culture. A common method involves using tubers with buds as explants, sterilizing and disinfecting them to induce adventitious buds. After proliferation, the resulting strong buds, seedlings, and rooting plants are cultured to obtain test-tube seedlings. With the completion of the genome sequencing of yellow calla lily, verification of the gene functions of colored calla lily urgently needs to be conducted. Establishing a callus-based regeneration system is a prerequisite for carrying out this research.

[0003] ZL201910992841.8 provides a method for inducing callus formation and establishing a regeneration system using colored calla lily bulbs as explants. However, it suffers from problems such as a long induction period and a low induction rate.

[0004] Finding a method to establish a colored calla lily regeneration system based on calla tissue with high induction rate and short induction cycle has always been one of the research directions of experts in this field.

[0005] This invention is hereby proposed. Summary of the Invention

[0006] To address the shortcomings and defects of existing technologies, one of the objectives of this invention is to provide a culture medium combination for constructing a regeneration system for colored calla lilies using tubers. This culture medium combination is based on a callus regeneration system, and the culture medium combination provided by this invention, when used with corresponding explants, can shorten the callus induction cycle and improve callus induction efficiency.

[0007] The second objective of this invention is to provide a method for constructing a regeneration system for colored calla lilies using tubers. This method uses non-budding tubers of colored calla lilies that have grown to a specific size as explants, induces the production of callus tissue, and establishes its regeneration system through proliferation culture and differentiation. This provides technical support for the establishment of a genetic transformation system for colored calla lilies and the large-scale production of seedlings. In particular, the establishment of a regeneration system for colored calla lilies provides technical support for the establishment of an Agrobacterium-mediated genetic transformation system.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of this invention provides a culture medium composition for constructing a colored calla lily regeneration system using tubers, the culture medium composition comprising:

[0010] Callus induction medium: MS-based medium, also containing the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or MS-based medium, also containing the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L;

[0011] Callus proliferation medium: MS as the basal medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L;

[0012] Differentiation medium: MS as the basal medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L.

[0013] Furthermore, the culture medium assembly also includes:

[0014] Shoot growth medium: MS as the basal medium, also containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, NAA 0.3-0.5 mg / L;

[0015] Furthermore, the culture medium assembly also includes:

[0016] Rooting medium: MS as the base medium, also containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, IBA 1.0-2.0 mg / L.

[0017] Preferably, the concentrations of 6-BA and NAA in the callus induction culture medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L.

[0018] Furthermore, the pH of the callus induction medium, the callus proliferation medium, the callus differentiation medium, the bud strengthening medium, and the rooting medium is all 5.75-5.85.

[0019] A second aspect of this invention provides a method for constructing a colored calla lily regeneration system using tubers, the method comprising the following steps:

[0020] S1 Selection and treatment of explants: Sterile colored calla lily seedling tubers that have completed bulb formation in bottles were selected as explants, with tuber diameters ranging from 0.8 to 1.2 cm; leaves and buds on the tubers were first removed, and then cut into small explant pieces for callus culture;

[0021] S2 Callus induction culture: The small explant pieces are inoculated onto a callus induction medium for induction culture, wherein the callus induction medium is based on MS medium and also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, based on MS medium and also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L;

[0022] S3 Callus proliferation culture: The small explants with callus tissue induced and cultured in step S2 were transferred to the callus proliferation culture medium for proliferation culture. The callus proliferation culture medium was MS as the base medium and also contained the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L.

[0023] S4. Differentiation culture of callus tissue: The callus tissue clumps obtained in step S3 are transferred to differentiation medium for shoot differentiation culture to obtain clustered shoots; the differentiation medium is MS as the base medium and also contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L;

[0024] S5 bud strengthening culture: The clustered buds are inoculated into a bud strengthening culture medium for bud strengthening culture;

[0025] S6 Rooting Culture: Rooting culture is carried out on rootless seedlings obtained after strong bud culture.

[0026] Furthermore, the size of the small explant piece is: 0.3-0.4 cm in length and width, and 0.1-0.3 cm in thickness;

[0027] And / or, the conditions for the induction culture are: dark culture for 30-40 days;

[0028] And / or, the conditions for the proliferation culture are: dark culture for 35-40 days;

[0029] And / or, after the proliferation culture, callus masses with a diameter of 1.0-1.5 cm are obtained;

[0030] And / or, the conditions for the bud differentiation culture are: alternating light and dark culture, wherein the light intensity is 800-1200 Lx, the daily light duration is 15-17 h, and the culture temperature is 25±1℃;

[0031] And / or, the bud differentiation culture time is 30-40 days;

[0032] And / or, the variety of the colored calla lily is Black Hole or Jingcai Sunshine.

[0033] Furthermore, the bud-strengthening medium is based on MS medium and also contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and NAA 0.3-0.5 mg / L;

[0034] And / or, the rooting medium is based on MS medium and also contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

[0035] Furthermore, the rootless seedlings have a height of 5.0-7.0 cm and a tuber diameter of 0.5-0.8 cm;

[0036] And / or, the conditions for the cultivation of vigorous shoots are: cultivation temperature 25±1℃, light and dark alternating cultivation, wherein the light intensity is 800-1200Lx and the daily light duration is 15-17h;

[0037] And / or, the time for cultivating the vigorous shoots is 30-35 days;

[0038] And / or, the conditions for the rooting culture are: culture temperature 25±1℃, light and dark alternation culture, wherein the light intensity is 800-1200Lx and the light duration is 15-17h per day;

[0039] And / or, the rooting culture time is 30-35 days;

[0040] And / or, the test-tube seedlings obtained after rooting culture are 6.0-10.0 cm in height, have 5-7 roots, and have a tuber diameter of 0.5-0.8 cm.

[0041] Furthermore, the method also includes:

[0042] Step S7 Seedling hardening and transplanting: After hardening the test-tube seedlings obtained from rooting culture, transplant them into the cultivation substrate for cultivation. Preferably, the cultivation substrate includes peat moss, perlite, and garden soil, wherein the mass ratio of peat moss:perlite:garden soil is 2:2:1.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] First, this invention uses sterile, budless tubers of colored calla lilies that have grown to a specific stage (e.g., tuber diameter of about 1.0 cm) as explants, and combines them with specific callus induction culture medium, proliferation culture medium and differentiation culture medium to obtain clustered shoots, and establishes its regeneration system. The establishment of this regeneration system provides technical support for the rapid propagation of colored calla lilies by tissue culture and the establishment of genetic transformation systems, especially for the establishment of Agrobacterium-mediated genetic transformation systems.

[0045] Secondly, the method of this invention can improve the induction rate of callus tissue and accelerate the formation of callus tissue. Generally, after about 20 days of induction culture, pale yellow, frosty callus tissue attached to the explant can be seen. Moreover, the buds differentiated from the callus tissue are relatively strong. Rooting culture can be carried out without the need for strong buds or with only one strong bud cycle, thereby completing the cultivation of tissue culture seedlings, shortening the seedling cycle, saving seedling costs, and greatly advancing the corresponding molecular biology research process. Attached Figure Description

[0046] Figure 1 Images of callus induction in the calla lily variety 'Jingcai Yangguang', where (a) is a photo taken on day 1 after the explants were transferred into the callus induction medium, and (b) is a photo taken on day 10 after the explants were transferred into the callus induction medium.

[0047] Figure 2 Image of callus mass obtained after callus tissue of callus cultivar 'Jingcai Yangguang' was transferred into proliferation medium;

[0048] Figure 3 A photo of clustered buds differentiated from callus tissue of the calla lily variety 'Jingcai Yangguang'. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] The embodiments of the present invention are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and processes are given. However, the protection scope of the present invention is not limited to the following embodiments. The process parameters in the following embodiments that do not specify specific conditions are generally in accordance with conventional conditions.

[0051] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0052] Unless otherwise specified, the process parameters in the following examples are generally performed under conventional conditions. All raw materials described in the following examples are available from publicly available commercial sources.

[0053] The first aspect of this invention provides a culture medium composition for constructing a colored calla lily regeneration system using tubers, comprising:

[0054] Callus induction medium: MS-based medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or MS-based medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L.

[0055] Callus proliferation medium: MS as the basal medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L;

[0056] Differentiation medium: MS as the basal medium, also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L.

[0057] The culture medium combination of this invention is screened for specific explants. Using sterile seedling tubers that have completed in-bottle heading as explants, and employing the above-mentioned callus induction medium, proliferation medium, and differentiation medium, callus induction can be highly efficient, with a high induction rate, rapid callus formation, and robust clustered shoots. The concentration of 6-BA in the callus induction medium should not be too high, as this will reduce the callus formation rate and even decrease the induction rate. The concentration of 6-BA should also not be too low, as this usually leads to a low induction rate. The concentration of NAA also needs to be compatible with the 6-BA concentration; otherwise, the induction rate will be low. An NAA concentration below 0.5 mg / L results in an induction rate below 40%. Replacing the 6-BA of this invention with other cytokinins such as KT, or replacing the NAA of this invention with other auxins such as 2,4-D, will not result in callus formation on the explant surface. Preferably, the concentrations of 6-BA and NAA in the callus induction medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L. This preferred callus induction medium can achieve an induction rate of over 70%.

[0058] In the regeneration system of the present invention, it is not suitable to add auxin-like substances to the proliferation medium, nor is it suitable to replace cytokinin 6-BA with other types of cytokinins. Adding auxin or replacing cytokinin 6-BA of the present invention with other cytokinins will result in a significant decrease in the proliferation coefficient.

[0059] In the regeneration system of the present invention, it is not suitable to add auxin-like substances to the differentiation medium, as adding auxin will lead to a decrease in the differentiation coefficient; in addition, the concentration of 6-BA should not be too high or too low, otherwise it will reduce the differentiation coefficient.

[0060] In the regeneration system of the present invention, it is not suitable to add cytokinin to the bud-strengthening culture medium, as adding cytokinin will reduce the effect of NAA.

[0061] In the regeneration system of the present invention, for the rooting medium, the auxin IBA has a better rooting effect than other auxins, while the addition of 6-BA to the rooting medium will reduce the rooting rate.

[0062] After the shoot clusters differentiate from the differentiation medium, it is necessary to strengthen the shoots and promote root growth, or directly strengthen the shoots. The preferred formulation of the shoot strengthening medium in the medium combination described in this invention is: MS as the base medium, and also containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, NAA 0.3-0.5 mg / L.

[0063] The preferred formulation of the rooting medium of the culture medium combination described in this invention is: MS as the base medium, and also containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

[0064] To ensure the cultivation effect, the pH of the callus induction medium, the callus proliferation medium, the callus differentiation medium, the differentiation medium, and the rooting medium are all 5.75-5.85.

[0065] A second aspect of this invention provides a method for constructing a colored calla lily regeneration system using tubers, comprising the following steps:

[0066] S1 Selection and treatment of explants: Select the tubers of sterile calla lily seedlings that have completed in-bottle formation as explants, wherein the diameter of the tubers is 0.8-1.2cm; first remove the leaves and buds on the tubers, and then cut them into small pieces for callus culture; in the example of the present invention, the size of the small pieces of explants is: length and width 0.3-0.4cm, thickness 0.1-0.3cm; the method for obtaining the tubers of sterile calla lily seedlings that have completed in-bottle formation can adopt the existing technology for rapid propagation of colored calla lily tissue culture, for example, refer to steps (1)-(3) of Example 1 in the patent document with publication number CN 112042532B.

[0067] S2 Callus induction culture: The small explant pieces are inoculated onto a callus induction medium for induction culture, wherein the callus induction medium is based on MS medium and also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or based on MS medium and also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L; In the example of the present invention, the induction culture conditions are: dark culture for 30-40 days;

[0068] Specifically, sterile seedlings with tubers (0.8-1.2 cm in diameter) are placed on an inoculation tray. All leaves and buds are removed, and the tubers are longitudinally cut into small pieces, 0.3-0.4 cm long and wide, and about 0.2 cm thick. These pieces are then inoculated onto callus induction medium and cultured in the dark. After about 20 days, most explants germinate and form a layer of pale yellow, frosty callus on their surface. With continued culture, the callus thickens. The selection of explants is closely related to the culture medium of this invention. The culture medium combination of this invention is suitable for using tubers that have grown to a size of 0.8-1.2 cm as explants; tubers that are too small or too large are not suitable for the culture medium of this invention. In particular, when tubers grow too large, cell differentiation is more complete, making dedifferentiation less likely, and the culture period is also longer. A growth of about 1 cm is most suitable.

[0069] S3. Callus proliferation culture: The small explants with callus tissue induced and cultured in step S2 are transferred to a callus proliferation culture medium for proliferation culture. The callus proliferation culture medium is MS-based and contains the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, and 6-BA 3.0-4.0 mg / L. In the example of this invention, callus clumps with a diameter of 1.0-1.5 cm are obtained after the proliferation culture. The proliferation culture conditions are: dark culture for 35-40 days.

[0070] Specifically, after 35-40 days of induction culture, the explants, along with the thickened callus, were transferred to a proliferation medium for further proliferation culture in the dark. After 35-40 days, the callus proliferated into clumps with a diameter of 1.0-1.5 cm.

[0071] S4. Callus Differentiation Culture: The callus clumps obtained in step S3 are transferred to differentiation medium for bud differentiation culture to obtain clustered buds. The differentiation medium is MS-based and contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, and 6-BA 1.0-2.0 mg / L. In this embodiment of the invention, the bud differentiation culture conditions are: alternating light and dark culture, with a light intensity of 800-1200 Lx, a daily light duration of 15-17 h, and a culture temperature of 25 ± 1 °C. The bud differentiation culture time is 30-40 days.

[0072] Specifically, callus clumps with a diameter of 1.0-1.5 cm were transferred to differentiation medium for bud differentiation culture. After 30-40 days of culture, the callus differentiated into clusters of buds.

[0073] S5 Bud Strengthening Culture: The clustered buds are inoculated into a bud strengthening culture medium for bud strengthening culture; the bud strengthening culture medium is preferably MS-based medium, and also contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, NAA 0.3-0.5 mg / L; in the example of the present invention, the bud strengthening culture conditions are: culture temperature 25±1℃, light and dark alternating culture, wherein the light intensity is 800-1200 Lx, and the daily light duration is 15-17 h; the height of the rootless seedlings is 5.0-7.0 cm, and the tuber diameter is 0.5-0.8 cm; the bud strengthening culture time is 30-35 days;

[0074] Specifically, the clustered buds are separated from the callus tissue and transferred to a bud-strengthening culture medium for bud cultivation. After 30-35 days of cultivation, the clustered buds grow into rootless seedlings with a height of 5.0-7.0 cm and a tuber diameter of 0.5-0.8 cm.

[0075] S6 Rooting Culture: Rootless seedlings obtained after bud strengthening culture are subjected to rooting culture. The rooting medium is preferably MS-based, containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L. In this embodiment of the invention, the rooting culture conditions are: culture temperature 25±1℃, alternating light and dark culture, with a light intensity of 800-1200 Lx and a daily light duration of 15-17 h; the rooting culture time is 30-35 days.

[0076] Specifically, the leaves of rootless seedlings with a height of 5.0-7.0cm are cut off, leaving only petioles about 1.0cm long, and then transferred to rooting medium for rooting culture. After 30-35 days of culture, they grow into test-tube seedlings with a height of 6.0-10.0cm, 5-7 roots, and tuber diameter of 0.5-0.8cm.

[0077] S7 Hardening and Transplanting: After hardening the test-tube seedlings obtained from rooting culture, transplant them into the cultivation substrate for further cultivation. Preferably, the cultivation substrate includes peat moss, perlite, and garden soil, wherein the mass ratio of peat moss:perlite:garden soil is 2:2:1.

[0078] Specifically, hardening-off is carried out indoors. After loosening the caps of the tissue culture bottles, place them in the buffer room of the tissue culture room for 2-3 days, then move them into the indoor room and place them under natural diffused light for another 2-3 days. After hardening-off, use tweezers to remove the test-tube seedlings, wash off the culture medium from the roots, and transplant them into 10×10 seedling trays. The cultivation substrate is peat moss: perlite: garden soil = 2:2:1. The cultivation substrate is sterilized with 800-1000 times diluted carbendazim before use, followed by normal water and fertilizer management.

[0079] The method of this invention is suitable for any existing calla lily variety, and is particularly suitable for cultivating the Jingcai Sunshine and Black Hole calla lily varieties, which are commercial colored calla lily varieties that can be purchased from flower markets or colored calla lily cultivation companies.

[0080] The following examples illustrate the establishment of regeneration systems for different varieties of calla lilies.

[0081] Example 1: Establishment of a regeneration system for the yellow calla lily variety 'Jingcai Yangguang'

[0082] (1) Explants were obtained by using tubers of 'Jingcai Yangguang' sterile seedlings that had completed bulb formation in the bottle. The tubers had grown to a diameter of about 1.0 cm.

[0083] (2) Callus Induction: Sterile seedlings with tubers approximately 1.0 cm long were placed on inoculation trays. All leaves and buds were removed. The tubers were longitudinally cut into small pieces approximately 0.3 cm long and wide, and 0.2 cm thick, and inoculated onto callus induction medium. The basal medium was MS, supplemented with 7.0 g / L agar, 30 g / L sucrose, 1.0 mg / L 6-BA, 0.8 mg / L NAA, pH 5.75-5.85, and cultured in the dark. After 3 weeks, the explants germinated and formed a layer of pale yellow, frosty callus on the surface. With continued culture, the callus thickened continuously. Figure 1 (a) is a photograph taken on day 1 after explants were transferred into the callus induction medium, and (b) is a photograph taken on day 10 after explants were transferred into the callus induction medium. On day 10, a light yellow, frost-like layer of callus tissue can be seen forming on the surface of a small number of explants. The callus induction rate in this example is shown in Table 1, number 16. A total of 50 small explant pieces were inoculated. The induction rate in Table 1 is a statistical analysis of the induction rate data after 30 days of callus induction culture of 50 small explant pieces. Successful callus induction was defined as the formation of a light yellow, frost-like substance on the surface of the small explant piece. The induction rate was calculated as (number of successfully induced samples / total number of samples 50) * 100%.

[0084] (3) Callus Proliferation: After 30 days of callus induction culture, 30 well-induced explants were transferred to proliferation medium for further culture. The basal medium was MS, supplemented with 6.7 g / L agar, 30 g / L sucrose, 3.0 mg / L 6-BA, pH 5.8, and cultured in the dark. After 30 days, the callus proliferated into clumps with a diameter of 1.2-1.5 cm. See [link to relevant documentation] for some of the callus clumps obtained after transfer to proliferation medium. Figure 2The callus proliferation fold in this embodiment is shown in the proliferation coefficient indicated by serial number 2 in Table 3. The proliferation coefficient in Table 3 is the result of weighing and counting after 30 days of proliferation culture. The sample size is 30. The proliferation fold = total weight of 30 samples after 30 days of callus proliferation culture / total weight of 30 samples before inoculation into the callus proliferation medium and after 30 days of callus induction culture.

[0085] (4) Callus Differentiation: Thirty callus clumps with a diameter of 1.2-1.5 cm were transferred to differentiation medium for shoot differentiation culture. The medium composition was: MS basal medium supplemented with 6.5 g / L agar, 30 g / L sucrose, 1.5 mg / L 6-BA, pH 5.8, light intensity of 800-1200 Lx, and a photoperiod of 16 h. After 30 days of culture, the callus differentiated into clusters of shoots. See the attached photographs for some of the callus that differentiated into clusters of shoots. Figure 3 The differentiation coefficient for this embodiment is shown in Table 4, number 3. The differentiation coefficient in Table 4 is calculated as: total number of shoots differentiated from all callus tissues / number of samples = 30.

[0086] (5) Bud Strengthening Culture: Thirty shoot clusters were isolated from the callus tissue and transferred to a bud strengthening culture medium for bud strengthening culture. The basic culture medium was MS, supplemented with 60 g / L sucrose, 7.0 g / L agar, and 0.5 mg / L NAA. The culture temperature was 25 ± 1℃, the light intensity was 800-1200 Lx, and the photoperiod was 16 h. After 35 days of culture, the shoot clusters grew into rootless seedlings with a height of about 5.0 cm and a tuber diameter of about 0.5 cm. The effect of the bud strengthening culture medium in this example is represented by the seedling height, specifically as shown in serial number 3 of Table 5. The seedling height in Table 5 = the sum of the seedling heights of all samples / the number of samples, 30.

[0087] (6) Rooting Culture: Leaves were removed from 30 rootless seedlings (approximately 5.0 cm in height), leaving only petioles about 0.5 cm long. These seedlings were then transferred to rooting medium for rooting culture. The basic medium was MS, supplemented with 50 g / L sucrose, 7.0 g / L agar, and 1.5 mg / L IBA. The culture temperature was 25 ± 1℃, the light intensity was 800-1200 Lx, and the photoperiod was 16 h. After 30 days of culture, the seedlings grew to a height of 8.0-10.0 cm, with 5-7 roots and a tuber diameter of 0.7 cm. The effectiveness of the rooting medium in this example was expressed by the rooting rate, specifically as shown in Serial No. 3 of Table 6. The rooting rate (%) in Table 6 is calculated as: number of rooted seedlings * 100 / number of inoculated seedlings. The number of rooted seedlings was calculated based on a seedling height of 8.0-10.0 cm and 5-7 roots.

[0088] (7) Hardening-off and Transplanting: Hardening-off was carried out indoors. After loosening the caps of the tissue culture bottles, they were placed in the buffer room of the tissue culture room for 3 days, and then moved into the indoor room and placed under natural diffused light for another 3 days. After hardening-off, the test-tube seedlings were removed with tweezers, the culture medium was washed off the roots, and they were transplanted into 10×10 seedling trays. The cultivation substrate was peat moss: perlite: garden soil = 2:2:1 (by weight), and the substrate was sterilized with 800-1000 times diluted carbendazim before use. Normal water and fertilizer management followed.

[0089] Based on Example 1, the inventors also conducted comparative experiments on various other culture medium formulations. The calla lily varieties, explant selection, and explant size on the induction medium were the same as in Example 1. The concentrations of agar and sucrose in the callus induction medium, proliferation medium, differentiation medium, shoot strengthening medium, and rooting medium were consistent with the corresponding concentrations in Example 1. The culture conditions and time for each stage were also the same as in Example 1, only the concentrations or types of auxin and cytokinin in each culture medium were adjusted. The calculation methods for the effect data of each stage of the culture medium were the same as those for the corresponding stage in Example 1, as detailed below:

[0090] (1) Callus induction under different callus induction media:

[0091] (1-1) Experiments with different concentrations of 6-BA and NAA: Two factors and four levels were set up, with 6-BA concentrations of 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2.0 mg / L and NAA concentrations of 0.3 mg / L, 0.5 mg / L, 0.8 mg / L, and 1.0 mg / L. An orthogonal design was performed, and the results are shown in Table 1 below. Among them, the combination of 6-BA 1.0 mg / L and NAA 0.8 mg / L, i.e., serial number 16, is the callus induction culture in Example 1 of this invention. Generally, a hormone combination with an induction rate higher than 60% is ideal for callus induction. As can be seen from the data in Table 1, when the hormone combination in the callus induction culture medium is 6-BA 1.0-2.0 mg / L and NAA 0.5-1.0 mg / L, the induction rate is above 46%.

[0092] Table 1. Effects of 6-BA and NAA concentrations on callus induction rate

[0093] Serial Number Number of explants 6-BA (mg / L) NAA (mg / L) Induction rate (%) 1 50 1.0 0.3 20.00 2 50 1.5 1.0 54.00 3 50 0.5 0.8 54.00 4 50 0.5 0.3 26.00 5 50 2.0 0.3 40.00 6 50 1.0 1.0 60.00 7 50 1.5 0.3 34.00 8 50 0.5 1.0 46.00 9 50 2.0 1.0 46.00 10 50 1.5 0.8 70.00 11 50 1.0 0.5 56.00 12 50 0.5 0.5 40.00 13 50 2.0 0.8 72.00 14 50 2.0 0.5 56.00 15 50 1.5 0.5 66.00 16 50 1.0 0.8 86.00

[0094] (1-2) Experiments on different combinations of cytokinins and auxins. The cytokinins selected were 6-BA or KT, and the auxin selected was 2,4-D to replace the NAA of the present invention. For detailed combinations, concentrations and induction rates, please refer to Table 2.

[0095] Table 2. Effects of different types of hormones on callus induction rate

[0096] Serial Number Number of explants Cytokinin (mg / L) Auxin (mg / L) Induction rate (%) 1 50 BA1.0 2,4-D1.0 0 2 50 KT 0.05 2,4-D 3.0 0 3 50 KT0.05 NAA0.5 0 4 50 0 2,4-D 3.0 0 5 50 0 2,4-D 2.0+NAA0.2 0

[0097] As shown in Table 2, the selection of cytokinins and auxins plays a key role in the induction of callus in the explants of this invention. Replacing auxin with 2,4-D or partially replacing NAA with 2,4-D will result in the induction of callus.

[0098] (2) Proliferation culture under different callus proliferation media: Proliferation media with different concentrations of 6-BA were set up, and a control experiment with added auxin NAA was also set up. The concentrations of 6-BA and NAA in the proliferation media of each group of experimental cases are shown in Table 3. The culture medium formula shown in serial number 2 is the proliferation culture medium formula of Example 1. The proliferation coefficient of the proliferation media of each group of experiments are shown in Table 3.

[0099] Table 3. Effects of different concentrations of 6-BA and NAA on callus proliferation.

[0100]

[0101]

[0102] As can be seen from Table 3, a 6-BA concentration higher than 4 mg / L or lower than 3 mg / L will significantly reduce the proliferation coefficient. In addition, the addition of auxin NAA to the proliferation medium system of the present invention will lead to a poor proliferation effect.

[0103] (3) Differentiation culture under different differentiation media: Differentiation media with different concentrations of 6-BA were set up, and a control experiment with added auxin NAA was also set up. The concentrations of 6-BA and NAA in the differentiation media of each group of experimental cases are shown in Table 4. The culture medium formula shown in serial number 3 is the differentiation media formula of Example 1. The differentiation coefficients of the differentiation media of each group of experiments are shown in Table 4.

[0104] Table 4. Effects of 6-BA and NAA concentrations on callus differentiation

[0105] Serial Number Number of explants 6-BA concentration (mg / L) Auxin NAA (mg / L) Differentiation coefficient 1 30 0.5 0 8 2 30 1.0 0 30 3 30 1.5 0 45 4 30 2.0 0 48 5 30 2.5 0 13 6 30 0.5 0.5 4 7 30 1.0 0.5 8 8 30 1.5 0.5 5 9 30 2.0 0.5 7 10 30 2.5 0.5 11

[0106] As can be seen from Table 4, a 6-BA concentration higher than 2 mg / L or lower than 1 mg / L will significantly reduce the differentiation coefficient. In addition, the addition of auxin NAA to the differentiation medium system of the present invention will lead to a poorer differentiation effect.

[0107] (4) Shoot cultivation under different shoot-enhancing media: Shoot-enhancing media with different concentrations of auxin NAA were set up, and a control experiment was also set up to determine whether or not 6-BA was added. The concentrations of 6-BA and NAA in the shoot-enhancing media of each group of experiments are shown in Table 5. Among them, the culture medium formula shown in serial number 3 is the shoot-enhancing culture medium formula of Example 1. The seedling height of the shoot-enhancing media of each group of experiments are shown in Table 5.

[0108] Table 5. Effects of 6-BA and NAA concentrations on shoot vigor.

[0109]

[0110]

[0111] As shown in Table 5, adding auxin NAA alone and controlling its concentration at 0.3-0.5 mg / L is beneficial for strong buds, and the seedling height is significantly higher than that of the control experiment with other concentrations. In addition, adding cytokinin 6-BA to this bud-strengthening culture medium system will significantly reduce the seedling height.

[0112] (5) Rooting culture under different rooting media: Rooting media with different auxins and with or without 6-BA were prepared. The concentrations of IBA, NAA, and 6-BA in the rooting media of each group of experimental cases are shown in Table 6. The culture medium formula shown in serial number 3 is the rooting culture medium formula of Example 1. The rooting rate of the rooting media of each group of experiments is shown in Table 6.

[0113] Table 6. Effects of IBA, NAA, and 6-BA on rooting

[0114]

[0115]

[0116] As shown in Table 6, replacing the auxin IBA in the rooting medium of this invention with NAA will significantly reduce the rooting rate; and the rooting rate will also decrease after adding 6-BA.

[0117] Example 2: Establishment of a Colored Calla Lily 'Black Hole' Regeneration System

[0118] (1) Explants were obtained by using the tubers of 'black hole' sterile seedlings that had completed bulb formation in the bottle. The tubers had grown to a diameter of about 1.0 cm.

[0119] (2) Callus Induction: Sterile seedlings with tubers approximately 1.0 cm long were placed on an inoculation tray. All leaves and buds were removed. The tubers were longitudinally cut into small pieces approximately 0.2 cm long, wide, and thick, and inoculated onto callus induction medium. The basal medium was MS, supplemented with 6.9 g / L agar, 30 g / L sucrose, 2.0 mg / L 6-BA, 0.5 mg / L NAA, pH 5.8, and cultured in the dark. After 23 days, the explants germinated and formed a pale yellow, frosty callus layer on the surface. With continued culture, the callus thickened. The callus induction rate was calculated using the same method as in Example 1; the induction rate in this example was 80%.

[0120] (3) Callus Proliferation: After 30 days of callus culture, the explants, along with the thickened callus, were transferred to proliferation medium for further culture. The basal medium was MS, supplemented with 6.7 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, pH 5.8, and cultured in the dark. After 30 days, the callus proliferated into clumps with a diameter of 1.0-1.3 cm. The fold increase was calculated using the same method as in Example 1; in this example, the fold increase was 4.5.

[0121] (4) Callus Differentiation: Callus masses with a diameter of 1.0-1.3 cm were transferred to differentiation medium for shoot differentiation culture. The medium composition was: MS basal medium supplemented with 6.5 g / L agar, 30 g / L sucrose, 2.0 mg / L 6-BA, pH 5.8, light intensity of 800-1200 Lx, and photoperiod of 16 h. After 35 days of culture, the callus differentiated into clusters of shoots. The differentiation coefficient was calculated using the same method as in Example 1, and the differentiation coefficient in this example was 48.

[0122] (5) Shoot Growth Culture: The clustered shoots were isolated from the callus tissue and transferred to a shoot growth culture medium for cultivation. The basic medium was MS, supplemented with 60 g / L sucrose, 7.0 g / L agar, and 0.3 mg / L NAA. The culture temperature was 25 ± 1℃, the light intensity was 800-1200 Lx, and the photoperiod was 16 h. After 32 days of cultivation, the clustered shoots grew into rootless seedlings with a height of approximately 7.0 cm and a tuber diameter of 0.6 cm. The seedling height was calculated using the same method as in Example 1, and in this example, the seedling height was 2.5 cm.

[0123] (6) Rooting Culture: Leaves were removed from rootless seedlings approximately 7.0 cm in height, leaving only petioles about 0.8 cm long. These seedlings were then transferred to rooting medium for rooting culture. The basic medium was MS, supplemented with 50 g / L sucrose, 7.0 g / L agar, and 2.0 mg / L IBA. The culture temperature was 25 ± 1℃, the light intensity was 800-1200 Lx, and the photoperiod was 16 h. After 30 days of culture, the seedlings grew to a height of 6.0-8.0 cm, with 5-6 roots and a tuber diameter of 0.6 cm. The rooting rate was calculated using the same method as in Example 1; the rooting rate in this example was 93.33%.

[0124] (7) Hardening-off and Transplanting: Hardening-off was carried out indoors. After loosening the caps of the tissue culture bottles, they were placed in the buffer room of the tissue culture room for 3 days, and then moved into the indoor room and placed under natural diffused light for another 3 days. After hardening-off, the test-tube seedlings were removed with tweezers, the culture medium was washed off the roots, and they were transplanted into 10×10 seedling trays. The cultivation substrate was peat moss: perlite: garden soil = 2:2:1 (by weight), and the substrate was sterilized with 800-1000 times diluted carbendazim before use. Normal water and fertilizer management followed.

[0125] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are within the scope of protection of the pending claims of the present invention.

Claims

1. A method for constructing a colored calla lily regeneration system using tubers, characterized in that, The method includes the following steps: S1 Selection and treatment of explants: Sterile colored calla lily seedling tubers that have completed bulb formation in bottles were selected as explants, with tuber diameters ranging from 0.8 to 1.2 cm. The leaves and buds on the tubers were first removed, and then the tubers were cut into small pieces for callus culture. S2 Callus induction culture: The small explant pieces are inoculated onto a callus induction medium for induction culture, wherein the callus induction medium is based on MS medium and contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, based on MS medium and contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L; S3 Callus proliferation culture: The small explants with callus tissue induced and cultured in step S2 were transferred to the callus proliferation culture medium for proliferation culture. The callus proliferation culture medium was MS as the base medium and also contained the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L. S4. Differentiation and culture of callus tissue: The callus tissue clumps obtained in step S3 were transferred to differentiation medium for shoot differentiation culture to obtain clustered shoots; the differentiation medium was MS basal medium, with agar 6.5-7.0 g / L, sucrose 30 g / L, and 6-BA 1.0-2.0 mg / L; S5 bud strengthening culture: The clustered buds are inoculated into a bud strengthening culture medium for bud strengthening culture; S6 Rooting Culture: Rooting culture is carried out on rootless seedlings obtained after strong bud culture.

2. The method according to claim 1, characterized in that, The size of the small explant piece is: 0.3-0.4 cm in length and width, and 0.1-0.3 cm in thickness; And / or, the conditions for the induction culture are: dark culture for 30-40 days; And / or, the conditions for the proliferation culture are: dark culture for 35-40 days; And / or, the conditions for the bud differentiation culture are: alternating light and dark culture, wherein the light intensity is 800-1200 Lx, the daily light duration is 15-17 h, and the culture temperature is 25±1℃; And / or, the bud differentiation culture time is 30-40 days.

3. The method according to claim 1, characterized in that, The bud-strengthening medium is based on MS medium and also contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and NAA 0.3-0.5 mg / L; And / or, the rooting culture is carried out using MS as the base medium, which also contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

4. The method according to claim 3, characterized in that, The conditions for the cultivation of vigorous shoots are: cultivation temperature 25±1℃, alternating light and dark cultivation, with light intensity of 800-1200Lx and daily light duration of 15-17 h; And / or, the time for cultivating the vigorous shoots is 30-35 days; And / or, the conditions for the rooting culture are: culture temperature 25±1℃, light and dark alternation culture, wherein the light intensity is 800-1200Lx and the light duration is 15-17 h per day; And / or, the rooting culture time is 30-35 days.

5. The method according to claim 1, characterized in that, The method further includes: Step S7 Hardening and Transplanting: After hardening the test-tube seedlings obtained from rooting culture, transplant them into the cultivation substrate for further cultivation.

6. The method according to claim 5, characterized in that, The cultivation substrate includes peat moss, perlite, and garden soil, wherein the mass ratio of peat moss:perlite:garden soil is 2:2:

1.

7. The method according to claim 1, characterized in that, The concentrations of 6-BA and NAA in the callus induction medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L.

8. The method according to claim 3, characterized in that, The pH of the callus induction medium, the callus proliferation medium, the differentiation medium, the bud strengthening medium, and the rooting medium is 5.75-5.85.

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